Difference between revisions of "Part:BBa K1887002"

Latest revision as of 17:41, 19 October 2016

NSR

Nisin is a 34-residue antibacterial peptide and a widely used food preservative because of its potent bactericidal activity against a variety of food spoilage bacteria. However, the bactericidal effect of nisin has been compromised by the occurrence of nisin resistance in various bacteria. Nisin resistance in the nisin nonproducer L. lactis subsp. diacetylactis DRC3 was reported to be conferred by a specific nisin resistance gene (nsr).

Sequence and Features

Molecular verification

We used PCR and restriction enzyme digestion to verify the NSR part on pSB1C3.

Background

The NICE system inducer, nisin, is a 34-residue food grade antibacterial peptide produced by some strains of L. lactis which is active against wide range of gram-positive bacteria and is widely used in the food industry as a preservative. Nisin inhibits growth of Gram-positive cells in two ways (Figure 2.1). First, nisin inhibits cell wall synthesis by binding to the lipid II molecule in the membrane. Second, nisin molecules can span across the lipid bilayer to make a pore in the cell membrane which causes the leakage of cytoplasm. The L. lactis NZ9000 strain has no resistant mechanism and cannot tolerate high levels of nisin concentration in the medium.

Antibacterial mechanisms of nisin

In nisin producing strains, the nisin operon contains four nisin immunity genes, nisI, nisF, nisE and nisG. Among them, nisI encodes a lipoprotein that serves as the first defense for nisin immunity (Figure 2.2A) In non-nisin-producing L. lactis strains, nisin resistance could be conferred by a specific nisin resistance (nsr) gene, which encodes a 35-kDa nisin resistance protein (NSR, Figure 2.2B). NSR could proteolytically inactivate nisin by removing six amino acids from the carboxyl “tail” of nisin. The truncated nisin (nisin 1–28) displayed a markedly reduced affinity for the cell membrane and showed significantly diminished pore-forming potency in the membrane. A 100-fold reduction of bactericidal activity was detected for nisin 1–28 in comparison to that for the intact nisin. What’s more, the truncated nisin can still induce gene expression through the NICE system. Thus, the incorporation of the nisI or nsr gene into the NZ9000 will lead to tolerance of NZ9000 at high levels of nisin concentration.

Nisin immunity protein NisI and nisin resistance protein NSR

Design

The NZ9000 or NZ-Blue strain (nisKR integrated) is sensitive to nisin. We inferred that the introduced of the nisI or nsr gene might render NZ9000 to tolerate high levels of nisin concentration while maintain the induced gene expression capacity of the NZ9000. To achieve the goal, we constructed two plasmids (Figure 2.3) to test whether the incorporation of nisI or nsr can really increase the nisin tolerance of NZ9000 and NZ-Blue. The expression of nisI or nsr gene is driven by the P32 promoter, a constitutive promoter.

Map of plasmid pMG36e-nsr (A) and pMG36e-nisI (B). Abbreviations: P32, promoter; nsr, nisin resistant gene; nisI, nisin immunity gene; Ter, terminator; Em^r, erythromycin resistant gene; Ori, replicon.

Results

The two plasmids were successfully constructed, and were transformed into NZ9000. After that we measure the growth curve of NZ9000, pMG36e-nisI/NZ9000 and pMG36e-nsr/NZ9000 under different concentrations of nisin. As shown in Figure 2.3, without nisin, the growth curve of these 3 strains exhibits no difference. However, under 200 IU/mL nisin concentration, the growth of NZ9000 is severely inhibited, the pMG36e-nisI/NZ9000strain can still grows to a moderate level, while the pMG36e-nisI/NZ9000 strain can still grow to a level similar to that without nisin, which means the NSR protein can protect L. lactis at high nisin concentrations, while the protect efficiency of NisI is not as pronounced as NSR. Finally, under 500 IU/mL nisin concentration, only the pMG36e-nsr/NZ9000 strain can still grow, the NZ9000 and pMG36e-nisI/NZ9000 strain cannot grow.

Growth Curve of NZ9000 (■), pMG36e-nisI/NZ9000 (△) and pMG36e-nsr/NZ9000 (◇) in different nisin concentrations. Abbreviations: IU, nisin international unit.

References

[1] Hacker, C., Christ, N.A., Duchardt-Ferner, E., Korn, S., Gobl, C., Berninger, L., et al. (2015). The Solution Structure of the Lantibiotic Immunity Protein NisI and Its Interactions with Nisin. J Biol Chem 290, 28869-28886.

[2] Khosa, S., Lagedroste, M., and Smits, S.H. (2016). Protein Defense Systems against the Lantibiotic Nisin: Function of the Immunity Protein NisI and the Resistance Protein NSR. Front Microbiol 7, 504.

[3] Sun, Z., Zhong, J., Liang, X., Liu, J., Chen, X., and Huan, L. (2009). Novel mechanism for nisin resistance via proteolytic degradation of nisin by the nisin resistance protein NSR. Antimicrob Agents Chemother 53, 1964-1973.